1. Field of the Invention
The present invention relates to a process for preparing lysophospholipid using enzyme, more specifically, a process for preparing lysophospholipid from glycerol-3-phosphate derivative using lipase based upon esterification or transesterification reaction.
2. Description of Prior Art
Phospholipids from natural sources contain several fatty acids, and their proportion depends on the source and fraction methods. For some practical applications, it is required to have phospholipids that contain specific fatty acid. In this case, chemical synthesis of phospholipid may be a promising approach. However, chemical methods for preparing phospholipids have many drawbacks. Very toxic and expensive solvents being employed should be removed, especially if the products are intended for food or pharmaceutical use. Further, substrates should be protected, deprotected and/or modified for preparing stereospecific phospholipid.
Lysophospholipid is the deacylated phospholipid at sn-1 or sn-2 position of glycerol backbone. Lysophospholipid is a surface active agent and used as emulsifier of food or cosmetics due to its high safety (Sarney, D. B., Fregapane, G., and Vulfson, E. N J. Ant. Oil. Chenz. Soc. 1994, 71, 93; Palta, J. P. and Farag, K. M. U.S. Pat. No. 5,126,155, 1992). Recently, lots of researches as to its biological properties have been carried out to apply this lysophospholipid to medical use (Buckalew, J. V. and Rauch, A. L. U.S. Pat. No. 4,7466,52, 1988).
Chemical synthesis of lysophospholipids requires not a few steps for protecting and deprotecting the substrate. Slotboom et. al. (Slotboom ,A. J., de Haas, G. H., and van Deenen, L. L. M. Chem. Phys. Lipids 1967 1, 317) prepared rac-1-stearyl lysophospholipid starting rac-1-stearyl-2-benzyl-3-iododeoxyglycerol. They used benzyl or trityl groups to protect the free hydroxyl group of glycerol and the blocking group was removed by the hydrogenolysis. This process was very complex and undesirable side-products were formed. Otherwise, the phospholipid having two fatty acids esterified with two hydroxyl groups has been prepared as by-product. Therefore, many attempts have been carried out to prepare lysophospholipid in biological method. The desired phospholipid with specific fatty acid which synthesized by chemical method or obtained by the fractionation of natural phospholipids were hydrolyzed by phospholipase A2 to corresponding lysophospholipid.
In the case of lysophophatidic acid (LPA) synthesis, it was more complex. LPA had to be prepared enzymatically either from the hydrolysis of lysopholipid by phospholipase D, or from the hydrolysis of phosphatidic acid by phosphatidic acid specific phospholipase A2 (Van Corven, E J., Van Rijswijk, A., Jalink, K., Van Der Bend, R. L., Van Blitterijk, W. J., and Moolenaar, W. H. Biochein. J. 1992, 281. 163). Calcium ion is required as a cofactor and should be controlled for efficient phospholipase A2 reaction.
Many lipase (EC-3.1.1.3.) have broad substrate specificity. Although natural substrates for the lipases are triglycerides, many of these enzymes have been used for breaking and forming of ester bonds in a wide varieties of compounds. There have been many reports on the modifications of phospholipids by lipase. Svensson et. al. (Svensson ,I., Adlercreutz, P., and Mattiasson, B. Applied Microbiol. Biotechnol. 1990, 33, 255) and Yagi et. al.(Yagi, T., Nakanishi, T., Yoshizawa Y., and Fukui F. J. Fennent. Bioeng. 1990, 69,23) investigated the transesterification of phosphatidylcholine with lipase. The lysophospholipid synthesis by lipase in a continuous reactor by transesterification was reported by Sarney et al.(1990). The inventors reported that lysophosphatidic acid (LPA) could be synthesized from glycerol-3-phosohate(G-3-P) with free fatty acid by lipase-catalyzed esterification in a solvent-free system. (Han, J. J. and Rhee, J. S. Biotechnol. Lett. 1995, 17, 531). A method for the production of lysophospholipid is characterized by the esterification of 1-hydroxyl group of glycerophospholipid by microbial, plant or animal lipase. Preferably, 12-22 saturated or unsaturated fatty acid is used at a concentration of 0.2-5.0 mol, preferably 0.5-2.0 mol on 1 mol of glycerophospholipid. In both case, water content control was the one of most important factor on the synthesis yield.
The water level of the reaction system is an important factor because it affects the equilibrium of esterification reaction. Water is formed during biocatalytic esterification. An organic reaction mixture may be characterized by a single water activity (a.sub.w) value instead of water content or concentration. In the case of water being a reactant in the desired conversion or side reaction, a.sub.w reactant determines the water mass action effect on the position of equilibrium. Many reports confirmed that the continuous control of a.sub.w during biocatalysis in organic media can increase yield and reaction rate.
Kahn et al. (Kahn, S. A., Halling, J. P. and Bell, G. Enzyme Microb. Technol. 1990, 12, 453) adjusted the a.sub.w of headspace above the reaction medium by circulation of head space gas through a drying column. The aluminum oxide sensor was used for continuous monitoring and control of a.sub.w during the lipase-catalyzed esterification.: however, the sensor has many limitations in stability, sensitivity, and measurement range. Another a.sub.w control method is to perform the reaction in a vessel with saturated salt solution in contact with the reaction mixture via the gas phase, so that the saturated salt solution continuously absorbs and releases water vapor to keep the a.sub.w constant. Svensson et al. (1993) developed a unique method for an a.sub.w control. A saturated salt solution was slowly circulated inside a silicon tubing which was contacted with traction medium, so that water vapor can be transported through the wall of the tubing, and thus the a. in the reaction medium can thereby be continuously controlled. In this system, however, the transport rate and equilibrium through the tubing was very slow and the apparatus was somewhat complex.
Another way for the continuous a.sub.w control is the use of salt hydrate pair. It was reported that a salt hydrate pair can control the water level in the reaction mixture by taking up or releasing water as required to keep a constant a.sub.w condition during the reaction (Halling, J. P. Biothehnol. Tech. 1992, 6, 271). Each kind of salt hydrate pair has a typical a.sub.w. When a hydrated salt and its corresponding lower hydrate or anhydrous form are present together, ideal behavior implies a fixed equilibrium water vapor pressure and hence a constant a.sub.w, whatever the relative quantities of the two forms.
The present invention developed the biosynthetic process for preparing lysophospholipid using lipase in a convenient process without protecting substrate. we also controlled the a.sub.w of the reaction medium by using a salt hydrate pair in order to increase the reaction rate and yield.